Studies of enzymes on the Vitamin B12 pathway : mechanistic studies of Uroporphyrinogen III synthase and function analysis of four methylases

Abstract

Several plasmids were constructed to generate Uro'gen III synthase using different expression systems. The C-1 azafulvene intermediate in the reaction of Uro'gen III synthase was trapped using NH4CI and NaBH4 to generate AMB and C-1 methyl bilane, respectively. Several low temperature 1 3C-NMR experiments were performed on a millimolar scale at high pH levels to study the enzymatic reaction. No evidence of a new intermediate or enzyme bound species was found. Several analogs of HMB were incubated with Uro'gen III synthase and studied for their effect on enzyme activity. Analogs with a substitution at the C-19 position showed an inhibitory effect on the enzyme. Although blocked from forming Uro'gen III, they were capable of forming the C-1 azafulvene intermediate, the first step in the reaction. The NMe analogs were also inhibitors but were not able to form the azafulvene presumably due a conformational distortion introduced by the presence of the methyl group on the nitrogen atom. Analogs with modified side chain groups are usually substrates for the enzyme as was seen with the butyrate bilane. The extra -CH2 group on the propionate side chain of ring D resulted in a slower substrate with a lower binding affinity for the enzyme. Four methyltransferases were expressed, purified, and analyzed for function.